Extracellular matrices are complex molecular networks that not only provide mechanical support, but by serving as reservoirs for bioactive molecules control cell growth and differentiation. Knowledge of the protein motifs and molecular forces that drive matrix assembly are largely unknown. Since several inherited disorders are associated with matrix dysfunction, elucidating molecular mechanisms that are used to regulate the assembly of extracellular networks is important for developing a basic understanding of how matrices form in normal and diseased states. The Drosophila eggshell is a specialized extracellular matrix amenable to genetic, biochemical, and morphological analyses. Understanding the principles and temporal hierarchies of interactions used in its assembly will advance our knowledge of how secreted proteins assemble into functional molecular networks in vivo. Using an array of specific antibodies and mutants as tools we propose an in depth study of sV23, a vitelline membrane protein that is essential for the formation of a functional eggshell. Within the context of our studies on sV23 we will begin to explore interactions amongst other proteins within the vitelline membrane network. In the first aim, by testing mutant transgenes in an sV23 protein null genetic background we will determine features of sV23 that are required for its assembly into a large, precisely aligned, polymeric network. Building on preliminary findings, in the second aim we will determine which perturbations in the structure of sV23 are capable of inducing dose-dependent sterility in wild type females. In the third aim we will use endogenous disulfide cross-links and an array of biochemical techniques including two- dimensional gel electrophoresis, gel filtration chromatography, affinity chromatography, and mass spectrometry to determine the molecular complexity of the sV23 disulfide-linked network and identify its covalent binding partners. Complexes from wild type animals will be compared with complexes from transgenic animals expressing mutant versions of the sV23 gene. The information obtained in this study will not only identify molecular strategies used to orchestrate the assembly of complex extracellular structures but may provide the basis for devising new strategies for insect vector control, thereby reducing the transmission of human diseases. The variety of techniques used in the project will provide opportunities for 6-8 undergraduate students to learn many research skills while fully participating in a research project. This study will contribute to our understanding of the molecular mechanisms that underlie the assembly of extracellular structures in normal and diseased states. The information obtained in this study may also provide the basis for new strategies to control the reproduction of vectors of human diseases, such as the mosquito. [unreadable] [unreadable] [unreadable]